The present invention relates to .beta.-glucans detection reagents which are used as diagnostic agents for nosomycosis in the pharmaceutical field and to methods of detecting .beta.-glucans using the reagents.
Currently, it has been attempted to detect nosomycosis at an early stage by determining the concentration of .beta.-glucans in blood because fungi contain .beta.-glucans in their components (Journal issued by Ohsaka University, School of Medicine, 41 (3): 133-142). Reagents specific to .beta.-glucans are therefore useful to detect nosomycosis and can be used as diagnostic agents in the pharmaceutical field.
It is known that the .beta.-glucans coagulate hemocyte lysate of horseshoe crabs, the finding that can be utilized to quantitatively determine levels of .beta.-glucans in blood. However, endotoxins also coagulate the hemocyte lysate of the horseshoe crabs. This is because the hemocyte lysate of the horseshoe crabs contain both an activator of the precursor of a .beta.-glucan coagulatory enzyme and an activator of the precursor of an endotoxin coagulatory enzyme.
The present inventors have successfully made reagents that specifically reacts with .beta.-glucans alone by removing or inactivating an activator of the precursor of an endotoxin coagulatory enzyme in the hemocyte lysate of a horseshoe crab, and filed this invention (Japanese Patent Application KOKAI Nos. 138193/1990, 207098/1990).
The invention disclosed in Japanese Patent Application KOKAI No. 138193/1990 relates to the hemocyte lysate specific to .beta.-glucans obtained by fractionating the hemocyte lysate by ion exchange chromatography using sulfopropyl as an adsorbent.
The invention disclosed in Japanese Patent Application KOKAI No. 207098/1990 relates to the hemocyte lysate specific to .beta.-glucans which comprises an endotoxin neutralizing peptide plus a limulus reagent used in the typical gelation procedure.
The present invention relates to the hemocyte lysate specific to .beta.-glucans obtained by extracting the hemocytes of American horseshoe crabs with 20 mM Tris-HCl buffer/pH 8.0 containing 12 mM MgCl.sub.2 and adding polyphemusin to the hemocyte lysate. The polyphemusin is obtained by extracting the precipitate of the hemocyte lysate with 20 mM HCl and purifying the resultant extract by reverse phase HPLC.
The hemocyte lysate specific to .beta.-glucans can detect as little as 0.1 ng/ml of carboxymethyl curdlan but does not react with as little as 100 ng/ml of the Nippon Pharmacopoeia standard endotoxin.
The method of the invention disclosed in Japanese Patent Application KOKAI No. 138193 is required to perform chromatography on endotoxin-free samples, which makes reagent preparing procedures complicated. In addition, although the method of preparing reagents disclosed in Japanese Patent Application KOKAI No. 138193/1990 is easy, a limulus reagent itself used in the gelation procedure is not suitable for quantitative analysis and its sensitivity is as low as 0.1 ng/ml.
It is an object of the present invention to provide a .beta.-glucans detection reagent which is easily prepared and very sensitive and a method of detecting .beta.-glucans using the reagent.
The present inventors have studied a reagent to detect .beta.-glucans, a reagent which is easily prepared and very sensitive, and found an easy method of preparing a .beta.-glucans detection reagent by combining typical endotoxin detection reagents with endotoxin neutralizing peptides derived from horseshoe crab hemocytes and chromogenic substrates.